Human serum albumin (HSA) is a single chain, non-glycosylated protein consisting of 585 amino acids, having a molecular weight of 66.5 kD and an isoelectric point between 4.7˜4.9. It is the most abundant protein in human blood plasma, making up about 60% of the total plasma proteins. There is about 40 g of HSA in per liter of human blood. Besides being present in the plasma, HSA is also found in tissues and body secretions, skins and lymph cavities. Under normal physiological conditions, HSA has an effect of maintaining plasma colloid osmotic pressure, nourishing, accelerating concrescence of wounds, and as a carrier, participating in transportation of many hydrophobic biological molecules such as hormones, biological active substances and drugs in the blood. Therefore, HSA is an important medical protein that is mainly used clinically for treatment of hypoproteinemia caused by loss of blood, burn, scald, plastic surgery and brain lesion, as well as for treatment of liver cirrhosis, hydronephrosis and so on. At present, HSA for clinical use is mainly prepared by extracting and isolating from human plasma. However, this preparation approach has the following disadvantages: on one hand, the source of plasma is insufficient, i.e. the limited blood supply is unable to meet the demands of production of HSA and the relevant preparations thereof; on the other hand, blood itself may potentially be a risk factor, for example it may contain dangerous infectious pathogens such as hepatitis virus, human immunodeficiency virus (HIV) and so on, which causes enormously concerns about the application of HSA extracted from plasma. Therefore, it is urgent to develop an alternative process to produce HSA.
With the development of modern DNA recombinant and synthesis techniques, researchers take a profound interest in the production and application of recombinant human serum albumin (rHSA). So far, various expression systems have been experimentally used for mass production of rHSA. For example, prokaryotes such as colon bacillus (Latta, M. et al., Bio/Technology, 5:1309-1314, (1987)), bacillus subtilis (Saunders, C. W. et al, J. Bacteriol. 169: 2917-2925, (1987)), eukaryotes such as yeasts (WO 00/44772, EP0683233A2, U.S. Pat. No. 5,612,196) and also cultivation of animal cells have been used for the production of rHSA. However, such approaches supra are not suitable for industrialized production either due to low expression level or high production cost. Chinese patent application No. 200510019084.4 of the present inventors discloses a method for producing rHSA using rice endosperm cells as bioreactor, comprising: using promoters and signal peptides specifically expressed in rice endosperm to mediate the entry of rHSA into endomembrane system of the endosperm cells of rice and store rHSA in the protein bodies of the rice endosperm, thus allowing rHSA to accumulate extensively in the rice grain and reach a higher expression level finally. The expression level of the obtained rHSA is at least above 0.3% based on the weight of the rice grain. The method has the advantages of high expression level and low cost, thereby it provides the possibility to develop a novel strategy for the production of protein drugs.
The rHSA produced by any expression system should be purified before entering market. The purification technique may affect the quality of the product as well as production cost. The cost of purification process makes up about 80˜90% of the total production cost. At present, there is no purification process for separating and purifying rHSA from rice grain. Therefore, it is technically difficult and economically risky to develop a simple and cost-effective purification process to purify rHSA from rice grain.
At present, the techniques for extracting rHSA from yeast and plant suspension cells have been reported. For example, Chinese patent application CN 101768206A disclosed a process for purifying rHSA expressed in Pichia pastoris, comprising: filtrating the fermentation broth of rHSA with a ceramic membrane, and sequentially subjecting the filtrate to cation exchange chromatography, hydrophobic chromatography and weak anion exchange chromatography to obtain purified rHSA. However, due to the substantial differences of the impurities among the rice grain, yeast and plant suspension cells, those prior art can not be directly used for separating and purifying rHSA from rice grain. Therefore, it is desirable to develop a simple and effective process for separating and purifying rHSA from rice grain to produce rHSA with high yield and high purity, which would provide a basis for future industrialized production.